Biochemical Changes Associated with Fresh-Cut Fruit Processing and

Chapter 4

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Biochemical Changes Associated with Fresh-Cut Fruit Processing and Storage Olusola Lamikanra and Michael A . Watson Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Robert E . Lee Boulevard, New Orleans, L A 70124

Fresh-cut fruit processing reduces product shelf life. Cut cantaloupe melon stored at 4 °C, unlike those kept at 20 °C, did not show significant changes in titratable acidity, pH, °Brix and organic acid content over a period of 14 days. Organic acids in the fresh fruit are oxalic, citric, malic and succinic acids. A t 20 °C, lactic acid was by far the dominant acid present after two days. Gram-positive mesophilic bacteria also grew rapidly at this temperature unlike the slow growing Gram-negative rods that were observed at 4 °C. Lactic acid and Gram-positive microflora are potential quality markers of temperature abuse during fresh-cut fruit storage. Peroxidase (POD) enzymes in cantaloupe, honeydew, kiwifruit, pineapple and watermelon are anionic ascorbate P O D apparently produced in response to wound induced oxidative stress. The P O D isozymes are stable for 20 min. at 50 °C, hence mild heat treatments for short periods of time will not reduce activity significantly. In the muskmelons, ascorbic acid is suggested to inhibit oxidation by complexing with P O D and metal ion cofactors.

52

© 2003 American Chemical Society

In Freshness and Shelf Life of Foods; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2002.


53 Fresh-cut produce is the fastest growing food category in the supermarket. Total sales have more than doubled from $5.2 billion in 1994 to an estimated $11 billion in 2000. Increased respiration rate, water and enzymatic activities that occur during fresh-cut fruit processing and storage cause the fruit tissue to senesce rapidly (1-4). Sugars, organic acids and amino acids significantly contribute to sweetness and aroma of fruits. Sweetness and aroma are the two most important quality indicators in melons (5,6). Organic acids are also important flavor precursors and respiratory energy sources in plant cells. Loss of fruit characteristic flavor is usually the first deteriorative change that occurs. Initial flavor changes are not indicated by appearance of the fruit. There is considerable interest in the factors and reactions that occur in the fruits to cause flavor changes. Degradative changes also include tissue softening/texture changes, nutrient losses, browning, and microbial growth. Cold storage generally retards many biochemical processes in foods, and fresh-cut fruits are normally stored under refrigeration. In the uncut fruit, enzymes are normally sequestered within the vacuole. Tissue disruption causes the mixture of the enzymes with other cytoplasmic and nucleic substrates. Changes that occur in P O D activity in wounded and fresh cut fruits significantly contribute to their product quality (7-9). In addition to the role played by P O D in flavor of fresh fruits and vegetables P O D isozymes are involved in many cell alterations (10). They appear to influence flesh firmness through catalysis of the cross-linking between tyrosine residues of the cell wall extensins and ferulic acid substituents of pectins (11), and the synthesis of lignin and suberin polymers (12). The mechanism of P O D peroxidative action essentially involves an oxidative action by way of an initial formation of a complex intermediate with a hydrogen acceptor. The transfer of hydrogen from a donor substrate results in a second complex intermediate before the regeneration of the P O D enzyme and formation of a reaction product (10). Plant PODs are classified into two types (13). Ascorbate P O D ( A P X ; class I), which is from the plant chloroplast and cystol, is distinguished from classical secretory plant P O D (guaiacol P O D ; class II) isozymes by significant differences in their primary structure. A P X is an H 0 -scavenging P O D that uses ascorbate as an electron donor in plants and algae (14,15). Most secretory plant P O D are glycosylated (16). Unlike guaiacol P O D that is characterized by broad specificity with respect to electron donors, and participates in many physiological processes such as the biosynthesis of lignin and ethylene, ascorbate P O D exhibits high sensitivity for ascorbate as the electron donor (14,17), and is specific in its physiological role in scavenging potentially harmful H 0 (18) and free radicals (19). Most of our ongoing research relates to the nature of P O D enzymes and how they potentially affect the quality and shelf life of fresh-cut fruits. The possible roles of P O D 2

2

2

2


54 enzymes in maintainingfreshness,and other biochemical effects on fresh-cut fruit quality are discussed.


Materials and Methods Determination of pH, Titratable Acidity and °Brix. Cantaloupe melons slices (~3mm thick) were prepared after skins and seeds were removedfromthe whole fruit. A set offruitpieces placed in plastic containers with airtight lids and kept in an incubator at 4 °C while the rest were stored at 20 °C inside a laminar flow hood. Juice obtained after centrifugation (5000 χ g) of pulverized fruit samples taken periodically were used for pH, soluble solids and titratable acidity determinations. Titratable acidity was determined as malic acid equivalents by titration with NaOH (0.1M). Organic Acid Analysis. Fruit slices were frozen in liquid nitrogen and lyophilized. The dried fruit was mixed with liquid nitrogen and crushed into powder in a mortar. Organic acids were extracted from the powder in acetic acid solution (pH 2.5) and analyzed by HPLC as previously described (20,21). Determination of Microbial Activities. Total microbial counts were carried out aerobically on Nutrient agar. Suspended homogenates (0.1 mL) of the fruit (1020 g) in sterile water (190 mL) were overlaid on the surface of Nutrient agar plate after series dilutions. Bacterial colonies were counted after 48 h of incubation at 30 °C. Effect of Ascorbic Acid on Cantaloupe Color During Storage. Cut fruit pieces (200 g) in polypropylene baskets and dipped in cold water (4 °C) and other solutions containing the following additives: ascorbic acid (1.25 mM), ascorbic acid (2.5 mM), ascorbic acid (2.2 mM) plus EDTA (10 mM) and ascorbic acid (2.5 mM) plus MnCl (2.5 mM) in separate treatments for 1 min. After the fruits were dipped in the respective solutions, they were allowed to drain for about 1 min before being transferred into sealed plastic containers. A set of fruit pieces that were not dipped in water was used as control. Treated fruits were stored at 4 °C for 20 days. Color changes determined from Tristimulus L*, a*, b* measurements on a HunterLab DP-9000 (Reston, VA) colorimeter were used to determine changes in L * (AL), Chroma (AC). 2

Peroxidase Enzyme Assays. Acetone powders were prepared by homogenizing cutfruits(200 g) with cold acetone (500 mL; -20 °C) for 30 s and successive washings offiltratewith cold acetone. In the preparation of watermelon acetone powder, water (10% w/w) was added to thefruitprior to addition of cold acetone and homogenization. Enzyme extractions were carried out by homogenizing


55 acetone powders (1 g) in a mixture of phosphate buffer (0.05 M ; p H 8.0,15 mL) and Triton X (0.1%) and eentrifugation of the homogenate. Peroxidase assay with guaiacol as substrate was carried by monitoring changes in absorbance at 470 nm in mixtures consisting of 0.02 M N a H P 0 and 0.8 M N a H P 0 , 2 0 m M guaiacol, 4 m M H 0 , enzyme extract (10 μΙ), p H 7, in a total volume of 3 mL (22). The effects of the following compounds: M n C l (0.5 m M ) , E D T A (10 mM) C a C l (0.5 mM), diethyldithiocarbamate (DETC; 0.5 mM), tiron (1 mM), superoxide dismutase (100 units/mL), ascorbic acid ( A A ; 0.5 mM), and β-mercaptoethanol (0.5 mM) on enzyme activity were determined. The compounds were added to the buffer at stated concentrations prior to preparation of reaction mixtures and enzyme activity assays. P O D activity as a function of pH was determined by adjusting p H of the buffer using suitable amounts of 1.0 M NaOH or 1.0 M HC1 containing 0.5 M N a C l . The effect of temperature on enzymatic activity was determined by incubating the reaction mixtures at different temperatures (30 - 70 °C) prior to addition of the enzyme, and measuring increase in absorbance at the corresponding temperatures. Thermal stability of cantaloupe melon peroxidase was determined by incubating enzyme extracts at 30 - 70 °C in a waterbath for 5 and 15 min at each temperature. Reaction mixtures were prepared at these time intervals and assayed for residual enzyme activities at each temperature. Peroxidase assay with A A as substrate was carried out in a buffer with a reaction mixture containing potassium phosphate (50 m M ; p H 7.0), ascorbate (0.5 m M ) , H 0 (0.1 mM) and E D T A (0.1 mM). Adding the enzyme extract started the reaction and the absorbance decrease was recorded 10 to 30 s after this addition (23). Enzymatic activities in the absence of H 0 were generally very weak or absent. Thus, correction for the oxidation of ascorbate as a result of ascorbate oxidase was unnecessary. 2

2

4

2

4

2

2


2

2

2

2

2

Gel electrophoresis. Electrophoretic separations were carried out on Xcell II Mini-Cell on 8cm χ 8cm χ 1mm, 10-well polyacrylamide pre-cast gels. Novex Tris-glycine buffer system was native gel electrophoresis. Isoelectric fcosing (pH 3-10) buffers were used with IEF gels for isoelectric focusing. Protein separations were performed according to the manufacturer's instructions (Novel Experimental Technology, San Diego, California). Peroxidase assay with benzidine was done by immersing gels in acidic benzidine (100 mL; 0.5% w/v benzidine and 4.5% w/v glacial acetic acid) for 5 min after which H 0 (3 m L of 30% v/v H 0 ) was added. Gels were photographed immediately. Staining with o-dianisidine was carried out by immersing gels in a solution containing sodium phosphate (10 m M , p H 6.0), o-dianisidine (1 mM) and H 0 (50 m M ) for 2 h at room temperature. 2

2

2

2

2

2


56 Results and Discussion


Cold storage (4 °C) helps to maintain a number o f cut fruit quality parameters. Titratable acidity, p H and soluble solid content did not change significantly over a period of two weeks in cut cantaloupe pieces stored at 4 °C (Table 1). Organic acid content and composition in the fruit were also stable over the same period of time (Table 2). Organic acids identified in fresh-cut and fruit stored at 4 °C are oxalic, citric, malic, and succinic acids. Table I: Changes in p H , total acidity (g/100 m L ) and soluble solids in minimally processed cantaloupe with storage at 20°C and 4°C. Storage Days

pH

Acidity

°Brix

4°C

0 1 2 3 5 14

6.47a* 6.42a 6.51a 6.51a 6.55a 6.30a

0.21a 0.20a 0.18a 0.26a 0.22a 0.19a

8.53a 8.60a 8.00a 7.87a 7.73a 7.37a

20 °C 0 1 2 3 5

6.58a 6.61a 5.99b 5.25b 4.60a

0.21e 0.25e 0.44d 0.83c 1.61b

9.03a 9.07a 6.60b 5.80b 4.74c

•Values in the same column for each temperature without the same letters are significantly different (Pcantaloupe>watermelon>pineapple=kiwifruit (Table 4). Reactivity in the presence of POD extracts from watermelon and pineapple with guaiacol, relative to enzymatic activity of POD extracts in the other fruits were higher than their corresponding order when A A was the substrate. The order of relative activity when guaiacol was the substrate was cantaloupe^atermelon>honeydew>pineapple>kiwifruit. (Table 4). Wounding, as in fresh-cut processing, generally causes an increase in ascorbate biosynthesis and ascorbate POD (36-39,44). Ascorbate contents in plant tissues are known to regulate ascorbate POD activity (40). Ascorbate POD production in response to oxidative stress could help reduce deteriorative reactions (45-47). In the fruits, POD production is obviously a part of the cell adaptation to stress


62 Table ΙΠ. Relative POD activity in 100g of fruit in the presence of guaiacol and H 0 (control), ethylenediaminetetraacetic acid (10 mM), tiron (1.0 mM), CaCl (0.5 mM), superoxide dismutase (100 units/mL), diethyldithiocarbamic acid (0.5 mM), 2-mercaptoethanol (0.5 mM), and ascorbic acid (0.5 mM). 2

2

2


POD Inhibitors

Fruit Tissue POD Activities Honeydew

Kiwifruit

Pineapple

Watermelon

Control

1.0a

1.0a

1.0a

1.0a

EDTA

0.84a

0.35b

0.82a

0.80a

Tiron

0.03b

0.01c

0.12b

0.04b

CaCl

0.82a

0.86a

0.89a

0.93a

2+

0.82a

0.42b

1.03a

0.98a

SOD

0.82a

0.98a

1.05a

0.90a

DETC

0

0

0

0

2-ME

0

0

0

0

Mn

2

0 AA 0 0 0 •Values in each row without the same letters are significantly different (P< 0.05).

Table IV. Relative POD activity in 100 g of fresh-cut fruit with A A and guaiacol substrates

POD Substrates

Relative POD Activity in Fruit Tissue Cantaloupe

Honeydew

Kiwifruit

Pineapple Watermelon

Ascorbic Acid

0.73b

LOOa

O.Old

0.02d

0.40c

Guaiacol

1.00a

0.76b

0.08d

0.51c

0.96a

•Values in each row without the same letters are significantly different (P< 0.05).



63 conditions created by surface tissue exposure. The P O D activities in the fruits could also be indicative of their relative fresh-cut oxidative stress activity, with cantaloupe melon having the highest stress level. When honeydew was dipped in A A solution, it caused a 42% decrease in P O D activity. The same was observed for cantaloupe melon (32). The effect of A A treatment in reducing fruit P O D activity was attributed to an ascorbate preservative role by way of an enhanced antioxidative action of the enzyme-hydrogen donor complex and trace metal ion cofactors either because of the antioxidant nature of molecular A A , or the utilization of P O D enzyme-hydrogen donor complex formation. Ascorbate treatment of fresh-cut kiwifruit and watermelon, however increased fruit P O D activity five and two fold respectively. Peroxidase activity in pineapple was unaffected by ascorbate treatment. It is unclear why the effects of ascorbate treatment on production of POD enzyme in the other fruits differ from those of the two muskmelon fruit. This could be related to the much higher activities of P O D from the muskmelons with A A substrate than enzyme extracts from the other fruits. The results, however, appear to indicate a product specific nature ascorbate-POD interactions in fruits, and the need for further research in this area. The optimum p H activity for kiwifruit P O D was considerably lower than those of the other fruits (Figure 3).



64

Figure 4. Effect of temperature on POD activity in honeydew (HD), kiwifruit (KI), pineapple (PI) and watermelon (WA).

Kiwifruit (pH 3.5) and pineapple (pH 4.5-5.5) both exhibited acidic optimum p H activities while that of watermelon (pH 6.5) was near neutral. Optimum p H activity for honeydew was the highest at p H 7.5. The P O D p H activity profile indicates that enzymatic activities in these fruits are optimum during their periods of normal harvest maturities. The more acidic kiwifruit and pineapple have their optimum activities at lower p H values than the relatively less acidic honeydew and watermelon. Honeydew and kiwifruit P O D were most active at 50 °C, and pineapple had an optimum, temperature activity at 60 °C (Figure 4). The optimum temperature for watermelon P O D activity was higher than those of the other fruits, occurring after 70 °C. Watermelon P O D was also the most stable after thermal treatments (30 to 70 °C) for 5 and 20 min. (Figure 5). Over 90% of enzymatic activity was retained after 20 min of incubation at 70 °C. Peroxidase stabilities in kiwifruit and honeydew followed a similar pattern. Activities, as illustrated by kiwifruit P O D stability illustrated in Figure 5, decreased significantly after incubation at temperatures above 50 °C for 20 min. P O D isozymes in pineapple were relatively stable up to 60 °C. The heat stability, p H and temperature activity optima for honeydew are quite similar to those of cantaloupe melon (22). Residual P O D after blanching and other forms of temperature treatments could be used as an index for enzyme inactivation because of their relative heat stability. The use of mild heat treatments for short periods of time (-50 °C for less than 5 min) as a minimal processing step for the cut fruits are not expected to affect P O D activity levels in the fruit significantly.



65

30

40

50

60

70

Temperature ( ° C ) Figure 5. Residual POD activity after thermal treatment of watermelon for 5 (— ·—) and 20 min ("" · ), and kiwifruit after 5 (— + — ) and 20 min. ( + ). Peroxidase catalysis of cell wall activities such as the cross-linking of ferulic acid substituents of pectin and the synthesis of lignin and suberin polymers are mainly ascribed to anionic isozymes (48). Six acidic P O D isozymes (pi 4.4 - 0 6.1) were separated by isoelectric focusing in pineapple while three were obtained for both honeydew and watermelon (Figure 6). The enzymes in honeydew (pi 5.5 - 6.0) and watermelon (pi 5.0 -5.8) are also acidic. Kiwifruit P O D enzyme was undetectable with benzidine hence it was not possible to determine its isoelectric property. The anionic P O D isozymes in the fruits would normally play significant roles in the firmness and texture of their fresh-cut products. Their role in the firmness and texture of fresh-cut fruits is, however, likely to be diminished by the preference of ascorbate P O D for ascorbate as the electron donor (17). Conclusions Appearance and quality parameters (titratable acidity, p H , soluble solids and organic acid composition) cannot be used as reliable determinants of the freshness of refrigerated fresh-cut fruits. While these parameters do not change appreciably with proper cold storage, enzymatic and microbial activities that affect quality continue. Quality changes that result from temperature abuse are, however, easier to detect.



66

Figureo: Benzidine stained isoelectric focusing ofPOD in honeydew (HD), kiwifruit (KI), pineapple (PI) and watermelon (WA).

The presence and amount of lactic acid and Gram-positive microflora could serve as indicators of temperature abuse during transportation and storage. In commonly fresh-cut processed fruits such as cantaloupe melon, honeydew, kiwifruit, pineapple and watermelon, P O D isozymes are anionic ascorbate P O D enzymes. Although anionic POD isozymes are involved in a number of cell wall activities, their effects on firmness and texture of fresh-cut fruits is likely to be diminished by the preference of ascorbate P O D for ascorbate as the electron donor. The enzymes appear to be produced as a response to fresh-cut processing induced oxidative stress. Ascorbic acid enhances the antioxidative action that involves metal ion cofactors in muskmelons apparently through ascorbate-POD complex formation. The nature of ascorbate-POD interactions in kiwifruit, pineapple and watermelon is unclear. Peroxidase activity profiles


67 indicate that enzymatic activities in the fruits are optimum during their normal harvest periods. M i l d heat (minimal processing treatments) for short periods of time will have no effect on POD activity.


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Biochemical Changes Associated with Fresh-Cut Fruit Processing and

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